Localized infection is a frequent complication of chronic wounds that interferes with the healing process and can lead to systemic infection, limb amputation, or death. Compounding the problem is an inability of the clinician to accurately detect localized infection by clinical observation, which is still the primary diagnostic method. This is because the usual signs of infection are masked by inflammation or the patients' underlying conditions or disease. The main objective of the proposed studies is to develop and validate a new assay that will enhance the ability of clinicians to detect chronic wound infection earlier and with more accuracy. The long-term goal is to incorporate this assay into a simple, inexpensive, point-of-care device to aid in diagnosis during a patient visit. This device will help individuals with chronic wounds receive appropriate treatment sooner, allow healing to resume, relieve their pain and discomfort, and avoid the spread of infection. The diagnostic device will be based on a novel detection method for pathogenic bacteria that was developed at ECI Biotech. This method measures the activity of protein-degrading enzymes (proteases) that infection-causing pathogens release into wound fluid. Since proteases are specific toward particular sites within proteins, ECI was able to design a single target peptide that contains cleavage sites for proteases from the most common wound pathogens. The resulting assay will detect the total number of pathogens but not discern between species. In previous studies, roughly 80% of clinical isolates from chronic wounds produced proteases in vitro that cleaved this target peptide. Also, over 90% of clinical samples taken directly from infected wounds were shown to have significant protease activity toward the target peptide. This work also revealed some cross-reactivity of the target peptide with the human neutrophil elastase (HNE), a host inflammatory protease present at high levels in chronic wounds. The first goal of this proposal is to alter the amino acid sequence of the target peptide to reduce interference by HNE while maintaining sensitivity toward bacterial proteases. The next goal is to demonstrate the efficacy of the new target peptide in detecting pathogens in chronic wounds. Protease activity in samples from patients with chronic wounds occupied by 105 or more pathogenic bacteria (infected) will be compared to those that are not (non-infected). In addition to verifying that this assay can differentiate infected from non- infected chronic wounds, this study will define a cutoff activity level that distinguishes the two. The last goal of our proposal is to correlate target peptide cleavage in vitro with known quantities of purified bacterial proteases. This information will be necessary to generate standards for conversion of proteolysis activity into a simple yes-or-no readout (visible by a color change) that will be used in the diagnostic device. Overall, the studies in this proposal will improve the assay for pathogen detection and demonstrate that it is suitable to aid in the early diagnosis of wound infection. /Relevance The goal of this research is to develop a simple, inexpensive diagnostic device to help clinicians determine if a chronic wound is infected at the point-of-care. By improving the ability for early detection of chronic wound infection, patients will receive appropriate treatment sooner and at a greater frequency. This will limit the spread of local wound infection, reduce the incidence of deep or systemic wound-related infections, and restore healing. [unreadable] [unreadable] [unreadable]